Immune stimulating dietary supplement and method of use thereof

ABSTRACT

The immune system of middle aged and elderly individuals is stimulated with a dietary supplement. The dietary supplement includes Vitamin E, Vitamin B6 and conjugated linoleic acid. The dietary supplement can further include glutathione alone or in combination with Vitamin C, folic acid, zinc, selenium, Vitamin D, copper and Vitamin B12. The dietary supplement is administered to middle aged and elderly individuals in a suitable form for consumption by the individual. Suitable forms of consumption can include a snack bar, tablet, capsule, powder, drink, or dairy products.

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional ApplicationSerial No. 60/133,101, filed May 7, 1999, the entire teachings of whichare incorporated herein by reference.

GOVERNMENT SUPPORT

This invention was made with Government support under 58-1950-9-001awarded by the United States Department of Agriculture. The Governmenthas certain rights in the invention.

BACKGROUND OF THE INVENTION

Immune responses gradually decline with increasing age. Coincident witha decline in immune responses is a concomitant increase in the incidenceof tumor development, infection and inflammatory diseases in middle agedand elderly populations of individuals. (“Fundamental Immunology” ed.W.E. Paul, Raven Press, NY (1989); Miller, R. A., Exp. Gerontol.29:21-35 (1994). Compromised nutritional status can contribute to theimpaired immunological state and, hence, declining health of agingindividuals. Thus, there is a need to develop convenient and effectivemethods that augment the nutritional requirements of middle aged andelderly individuals, thereby stimulating the immune system to combatdisease.

SUMMARY OF THE INVENTION

The present invention relates to a dietary supplement. It also isdirected to a method to stimulate the immune system of middle aged andelderly individuals or to stimulate proliferation of a lymphocyte byadministration of the dietary supplement.

In one embodiment, the dietary supplement comprises Vitamin E, VitaminB6 and conjugated linoleic acid. In a specific embodiment, the dietarysupplement includes Vitamin E in an amount in a range of between about10 milligrams and about 267 milligrams per milligram of Vitamin B6, andconjugated linoleic acid in an amount in a range of between about 17milligrams and about 100 milligrams per milligram of Vitamin B6. Inanother specific embodiment, the dietary supplement further includesglutathione Vitamin C, folic acid, zinc, selenium, Vitamin D, copper,Vitamin B12 and glutathione. Preferably, the dietary supplement includesVitamin C in an amount in a range of between about 17 milligrams andabout 200 milligrams per milligram of Vitamin B6; folic acid in anamount in a range of between about 0.05 milligrams and about 0.2milligrams per milligram of Vitamin B6; zinc in an amount in a range ofbetween about 1.67 milligrams and about 10 milligrams per milligram ofVitamin B6; selenium in an amount in a range of between about 0.005milligrams and about 0.02 milligrams per milligram of Vitamin B6;Vitamin D in an amount in a range of between about 0.0008 milligrams andabout 0.005 milligrams per milligram of Vitamin B6; copper in an amountin a range of between about 0.00008 milligrams and about 0.0007milligrams per milligram of Vitamin B6; Vitamin B12 in an amount in arange of between about 0.0002 milligrams and about 0.001 milligrams permilligram of Vitamin B6; and glutathione in an amount in a range ofbetween about 4 milligrams and about 33 milligrams per milligram ofVitamin B6.

in another embodiment, the dietary supplement comprises about 60-800 IUof Vitamin E (such as d-α-tocopherol, dl-α-tocopheryl acetate,dl-α-tocopheryl); about 3-6 mg of Vitamin B6; and about 100-300 mg ofconjugated linoleic acid. In another embodiment, the dietary supplementis administered to a middle aged or elderly individuals .

In another embodiment, the dietary supplement further includes about25-100 mg of glutathione (such as γ-glutamylcysteinylglycine) alone, orin combination with about 100-600 mg of Vitamin C; about 300-600 μg offolic acid; about 10-30 mg of zinc; about 30-60 μg of selenium; about5-15 μg of Vitamin D; about 0.5-2 μg of copper; and about 1-3 μg ofVitamin B12.

In yet another embodiment, the invention is a method of stimulating theimmune system of an individual comprising formulating the dietarysupplement and administering the dietary supplement to the individual.

In still another embodiment, the invention is a method of stimulatingthe proliferation of lymphocytes comprising formulating the dietarysupplement and administering the dietary supplement to the lymphocytes.In a preferred embodiment, T-lymphocytes (T-cells) are stimulated toproliferate.

The invention described herein provides an alternative and improveddietary supplement that can conveniently be used to stimulate immuneresponses in middle aged and elderly individuals. Thus, administrationof the dietary supplement of the present invention can potentiallyprevent infection-mediated diseases and tumor formation as a result ofcompromised immune function in individuals, in particular middle agedand elderly individuals.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates the effect of varying dilutions ({fraction (1/10)};⅕; {fraction (1/2.5)}; {fraction (1/1)}) of the nutrient mixture onConacavalin A-stimulated splenocyte proliferation in cells isolated fromaged C57BL/6 mice.

FIG. 2 depicts the body weight (grams) of young mice fed a control diet(young control - - - × - - - ), aged mice fed a control diet (agedcontrol - - - ∘ - - - ) and aged mice fed the nutrient mixture (agedtreatment - - -  - - - ).

FIG. 3 illustrates the effect of Conacavalin A on splenocyteproliferation in cells isolated from young and aged mice not fed thenutrient mixture (young control and aged control, respectively) and agedmice fed the nutrient mixture (aged nutrient). p<0.05 (*); p<0.0 1 (**).

FIG. 4 illustrates the effect of phytohemagglutinin (PHA) on splenocyteproliferation in cells isolated from young and aged mice not fed thenutrient mixture (young control and aged control, respectively) and agedmice fed the nutrient mixture (aged nutrient). p<0.05 (*); p<0.01 (**).

DETAILED DESCRIPTION OF THE INVENTION

The features and other details of the invention, either as steps of theinvention or as combinations of parts of the invention, will now be moreparticularly described and pointed out in the claims. It will beunderstood that the particular embodiments of the invention are shown byway of illustration and not as limitations of the invention. Theprinciple features of this invention can be employed in variousembodiments without departing from the scope of the invention.

The present invention relates to the discovery that a dietary supplementcan stimulate the immune system of middle aged and elderly individuals.In particular, the dietary supplement of the invention stimulates thefunction of and proliferation of T-lymphocytes (T-cells) of middle agedand elderly individuals. The phrase “dietary supplement” is usedinterchangeably herein with the phrase “nutrient mixture.”

In one embodiment, the dietary supplement comprises Vitamin E, VitaminB6 and conjugated linoleic acid. In a specific embodiment, the dietarysupplement includes Vitamin E in an amount in a range of between about10 milligrams and about 267 milligrams per milligram of Vitamin B6, andconjugated linoleic acid in an amount in a range of between about 17milligrams and about 100 milligrams per milligram of Vitamin B6. Inanother specific embodiment, the dietary supplement further includesglutathione Vitamin C, folic acid, zinc, selenium, Vitamin D, copper,Vitamin B12, and glutathione. Preferably, the dietary supplementincludes Vitamin C in an amount in a range of between about 17milligrams and about 200 milligrams per milligram of Vitamin B6; folicacid in an amount in a range of between about 0.05 milligrams and about0.2 milligrams per milligram of Vitamin B6; zinc in an amount in a rangeof between about 1.67 milligrams and about 10 milligrams per milligramof Vitamin B6; selenium in an amount in a range of between about 0.005milligrams and about 0.02 milligrams per milligram of, Vitamin B6;Vitamin D in an amount in a range of between about 0.0008 milligrams andabout 0.005 milligrams per milligram of Vitamin B6; copper in an amountin a range of between about 0.00008 milligrams and about 0.0007milligrams per milligram of Vitamin B6; Vitamin B12 in an amount in arange of between about 0.0002 milligrams and about 0.001 milligrams permilligram of Vitamin B6; and glutathione in an amount in a range ofbetween about 4 milligrams and about 33 milligrams per milligram ofVitamin B6.

in another specific embodiment, the dietary supplement comprises about60-800 IU of Vitamin E; about 3-6 mg of Vitamin B6; and about 100-300 mgof conjugated linoleic acid. In another embodiment, the dietarysupplement further includes about 25-100 mg of glutathione alone or incombination with about 100-600 mg of Vitamin C; about 300-600 μg offolic acid; about 10-30 mg of zinc; about 30-60 μg of selenium; about5-15 μg of Vitamin D; about 0.5-2 μg of copper; and about 1-3 μg ofVitamin B12.

The dietary supplement is formulated by blending the components (e.g.,Vitamin E, Vitamin B6, conjugated linoleic acid, glutathione) to producea mixture. In a particular embodiment, Vitamin E is d-α-tocopherol,dl-α-tocopheryl acetate or dl-α-tocopheryl and glutathione isγ-glutamylcysteinylglycine. Methods to mix the components of the dietarysupplement can include, for example, the addition of components ofrelatively lower concentration or quantity (e.g., selenium, copper) tocomponents of relatively higher concentration or quantity (e.g., VitaminC, conjugated linoleic acid, glutathione). The dietary supplement can bestored at ambient temperature (e.g., 18-25° C.) or at coolertemperatures, for example, in a refrigerator (e.g., 4° C.).

In one embodiment, the dietary supplement is formulated by blendingVitamin C, Vitamin B6, zinc, folic acid, selenium, copper, Vitamin D,Vitamin B12 and glutathione to prepare a stock mixture of the dietarysupplement. The fat soluble components, Vitamin E and conjugatedlinoleic acid, are then .added to the stock mixture prior to use (e.g.,in vitro addition to lymphocytes). In another embodiment the dietarysupplement is formulated by simultaneously blending all of thecomponents in any order.

As defined herein, a “middle aged” individual refers to an individualwho is at an age between about 40% to about 60% of the expected lifespanof the individual. For example, a middle-aged human individual is ahuman between the age of about 45 to about 65 years old. Likewise, amiddle-aged C57BL/6NCrlBr mouse is a mouse about 14 to about 16 monthsof age. An “elderly” individual refers to an individual who is an age ina range of between about 65% to about 100% of the expected lifespan ofthe individual. For example, an elderly human individual is a humangreater than about 65 years of age. Similarly, an elderly C57BL/6NCrlBrmouse is a mouse greater than about 16 months of age, such as a mouse ofabout 24 months of age.

The term “individual” is used to refer to any mammal. For example, themammal can be a primate mammal (e.g., human, monkey, baboon)domesticated mammal (e.g., dog, cat), ruminant mammal (e.g., cow, sheep,horse) or rodent mammal (e.g., mouse, rat, guinea pig). In particular,the dietary supplement is used to stimulate the immune system of humanmammals. It is also envisioned that the dietary supplement can be usedin veterinary practice to stimulate the immune system of other mammalssuch as dogs and cats.

In a method of the invention, the dietary supplements are administeredto middle aged and elderly individuals to stimulate the immune system.The supplement is administered to the individuals by a means suitable,for consumption, such as a tablet or capsule which can be easilyswallowed, chewed, or dissolved. Additionally, or alternatively, thedietary supplement can be formulated into a powder or liquid forconvenient addition to drinks, baked goods, dairy products or other foodstuffs. The dietary supplement can also be consumed in the form of asnack bar, drink or lozenge. The supplement preferably is consumed on adaily basis, e.g., once a day. The dietary supplement can also beconsumed multiple (greater than one) times a day.

The term “stimulate”, as used herein, refers to any augmentation,increase, or enhancement of an immunological indice in the individualfollowing consumption of the dietary supplement. In a preferredembodiment, the dietary supplement stimulates T-lymphocytes (alsoreferred to herein as T-cells) function. In a particularly preferredembodiment, T-lymhphocyte proliferation is the stimulated immuneresponse.

Optionally, stimulation of the immune system of the individual beingadministered the dietary supplement can be monitored. Methods to assessimmune system stimulation are routine and well known to those skilled inthe art. For example, indices of immune stimulation can be assessed inperipheral blood samples obtained from individuals consuming the dietarysupplement by monitoring the proliferation of phytohemagglutinin-(PHA)or concanavalin A(ConA)-stimulated lymphocytes. Additionally, oralternatively, the levels of immunoglobulins (e.g., IgA, IgG and IgM),antibody titers against known, vaccines or antigens, concentrations andsynthesis of cytokines (e.g., IL-2) and the number and type ofleucocytes (e.g., polymorphonuclear or mononuclear) can be determined inperipheral blood samples. Methods to assay immune stimulation can alsoinclude evaluations of delayed-type hyper-sensitivity skin responses,resistance to infections diseases, natural killer cell activity, or anycombination thereof. Exemplary techniques to assess immune systemstimulation can be found, for example, in Kennes, B., et al.,Gerontology 29:305-310 (1983); Meydani, S. N., et al., Mech. Aging andDevelop. 34:191-201, (1986); Meydani, S. N., et al., Ann. N.Y. Acad.Sci. 570:283-290, (1989); Meydani, S. N., et al., Am J. Clin, Nutr.52:557-563, (1990); Meydani, S. N., et al., Am. J Clin. Nutr.53:1275-1280 (1991); Wu, D., et al., J Nutr. 124:655-663, (1994);Meydani, S. N., et al., JAMA 277:1380-1386 (1997); Hayek, M; G., et al.,J Infect. Dis. 17:273-276 (1997), the teachings of all of which arehereby incorporated by reference in their entirety).

Immune responsive indices following treatment with the dietarysupplement of the invention can be compared with indices prior totreatment: If necessary, the dose and frequency of administration of thedietary supplement can be increased to further stimulate the immunesystem. Similarly, the dose and frequency of administration of thedietary supplement can be decreased following stimulation of the immunesystem (e.g., T-lymphocyte proliferation).

In another embodiment, the invention is a method of stimulating theproliferation of lymphocytes. The dietary supplement is formulated andadministered to the lymphocytes (e.g., T-lymphocytes, B-lymphocytes). Ina preferred embodiment, T lymphocytes are stimulated to proliferate.Stimulation of T-lymphocytes is a well recognized indice of T-lymphocytefunction, Beharka, A., et al., Methods in Enzymology 282:247-263 (1997);“Fundamental Immunlogy”, ed. W. E. Paul, Raven Press, NY (1989);“Methods in Cellular Immunology”, ed. Fernandez, R., et al., CRC Press,Boca Raton. Fla. (1995), the teachings of all of which are herebyincorporated by reference in their entirety. T-lymphocytes can bestimulated to proliferate by the methods of the invention, for example,by stimulating proliferation in a mammal in vivo following consumptionof the dietary supplement. Similarly, T-lymphocytes can be obtained fromthe mammal (e.g., from the spleen or peripheral blood) and exposed tothe dietary supplement in vitro to stimulate proliferation; thisstimulation can be an assessment of the effect of the dietary supplementon T-lymphocyte function in vivo. T-lymphocytes obtained from the mammalcan be a population or collection of cells which consist essentially ofT-lymphocytes, or a population of cells that consist of T-lymphocytes aswell as other cell types (e.g., B-lymphocytes, fibroblast, red bloodcells). Suitable methods to assess T-cell proliferation are well knownto one of skill in the art (See, for example, Wu, et al., J Nutr.128:193-197 (1998)), the teachings of which are hereby incorporated byreference in its entirety).

The present invention is further illustrated by the following examples,which are not intended to be limiting in anyway.

EXAMPLE 1 In Vitro Effects of the Nutrient Mixture on Cells Isolatedfrom aged Animals

Materials and Methods:

A stock mix for use in in vitro experiments was formulated by blendingthe components in the units and order as listed in Table 1. RPMI 1640media (Bio Whitaker, Walkerville, Md.) was used to prepare the stockmixture and for dilution of components added to the stock mixture(vitamin E and conjugated linoleic acid). The stock mixture was storedat ambient temperature (e.g., 18-25° C.). For in vitro experiments, thestock mixture was prepared fresh prior to dilution in RPMI 1640 media.

TABLE 1 Composition of the Stock Mixture for In Vitro ExperimentsComponent Units Vitamin C 500 mg Vitamin B6 5 mg Zinc 20 mg Folic Acid500 μg Selenium 50 μg Copper 14 μg Vitamin D 10 μg Vitamin B12 1 μgGlutathione 25 mg

The stock mixture was dissolved in RPMI 1640 media to prepare a solutioncontaining 12.5 milligrams of the stock mixture per milliliter of RPMI1640 media. For example, 62.5 mg of the stock mixture was dissolved in 5ml of RPMI 1640 media which was then serially diluted to prepare a 12.5microgram of the stock solution per milliliter of RPMI 1640. Theconcentrations of each component in a 12.5 μg/ml of the stock mixture inRPMI 1640 media are listed in Table 2.

A stock solution of conjugated linoleic acid (1mg/ml) was prepared bydissolving 25 μl of a 20 mg conjugated linoleic acid per ml stocksolution in 475 μl of RPMI 1640 media. A 2.5 μg/ml solution was thenprepared by adding 50 μl of the 1mg/ml solution to 19.95 ml of RPMI 1640media.

Vitamin E (d-α-tocopherol) was prepared as a 0.5 μg/ml solution.Initially a 2 mg/ml stock solution was prepared by dissolving 29.4 μl ofa d-α-tocopherol stock (34 mg/ml) into 471.6 μl of fetal bovine serum(FBS). Fetal bovine serum was used in these experiments since in vivovitamin E is incorporated into serum lipoproteins before it can bedelivered to cells. Fetal bovine serum contains lipoproteins, thereforeit is used as a vehicle to deliver vitamin E to cells in in vitrosplenocyte cultures. The vitamin E dissolved in fetal bovine serum wasincubated at 37° C. for fifteen minutes to allow incorporation ofvitamin E into the fetal bovine serum lipoproteins. Following theincubation, a 5 μg/ml solution of vitamin E was prepared by dissolving50 μl of a 2 mg/ml solution into 19.95 ml of RPMI 1640.

TABLE 2 Units of Each Component in a 12.5 μg/ml of the Stock Mixture forIn Vitro Experiments Component Units μg/ml Vitamin C 11.89 Vitamin B60.1189 Zinc 0.4756 Folic Acid 0.0119 Selenium 0.00119 Copper 0.000034Vitamin D 0.00024 Vitamin B12 0.000024 Glutathione 0.59460

The mixture listed in Table 2 was supplemented with conjugated linoleicacid and vitamin E so that the concentrations of each of the componentsin the nutrient mixture added to cells in vitro were as listed in Table3. For example, 5 ml of the mixture in Table 2 (12.5 μg/ml) was admixedwith 2.5 ml of vitamin E (5 μg/ml stock solution) and 2.5 ml ofconjugated linoleic acid (2.5 μg/ml stock solution).

TABLE 3 Concentration of Nutrients Added to Cells in In Vitro CulturesUnits (ng/nd) Added to Cell Component Units μg/ml Cultures (1 × 10⁶cells/ml) Vitamin C 5.945 2378 Vitamin B6 0.0594 23.76 Zinc 0.2378 95.12Folic Acid 0.00595 2.38 Selenium 0.000595 0.238 Copper 0.000017 0.0068Vitamin D 0.00012 0.048 Vitamin B12 0.000012 0.0048 Glutathione 0.297301118.92 Vitamin E 0.625 250 Conjugated linoleic acid 0.312 124.8

Aged (24 months of age) C57BL/6NCrlBR mice (n=6) were used in theseexperiments. The mean life span of a C57BL6 mouse is about 28 months.Aged mice were fed a commercial chow diet of Commercial Rodent Diet,Teklad 7012 (Teklad, Madison, Wis.).

Splenocytes were obtained from aged mice, cultured in the presence orabsence of the nutrient mixture and their ability to proliferate inresponse to the T cell mitogen, Con was determined as previouslydescribed (See, for example, Wu, et al., J Nutr. 128:193-197 (1998), theteachings of which are hereby incorporated by reference in theirentirety). Briefly, mice were euthanized with CO₂ asphyxiation, andtheir spleens were removed aseptically and placed in sterile RPMI 1640media (Bio Whitaker, Walkerville, Md.) supplemented with 25 mM HEPES/L,2mM glutamine/L, 1×10⁵ units penicillin/L and 100 mg streptomycin/L.

A 100 μl aliquot of the mixture in the concentrations listed in Table 3was added to 100 μl aliquot of splenocyte cell cultures (2.5×10⁶ mlcells) and incubated for four hours. The nutrient mixture was diluted1/1; 1/2.5; 1/5 and 1/10 (volume:volume) with RPMI 1640 culture medium.

After the four hour incubation (37° C., 5% CO₂ and 95% humidity), Con A(1.5 mg/L of RPMI 1640 medium) was added to splenocyte cultures. Con Aand PHA only stimulate T cells to proliferate (“Fundamental Immunology”,ed. W. E. Paul, Raven Press, NY (1989); “Methods in CellularImmunology”, eds. Fernandez, R., et al., CRC Press, Boca Raton, Fla.(1995), the teachings of both of which are hereby incorporated byreference in their entirety)., Therefore, the proliferative response toCon A and PHA is referred to as a T-cell mediated response. The cellswere cultured for an additional 72 hours (37° C.). Con A and PHA wereused in these experiments because lymphocytes from aged mice and humanshave been shown to have a significantly lower proliferative response tothe T cell mitogens ConA and PHA (Miller, R. A., Exp. Gerontol. 29:21-35(1994), the teachings of which are hereby incorporated by reference inits entirety). In addition, a mitogenic response induced by ConA is usedas a measure of T cell function (Beharka, A., et al., Methods itsEnzymology 282:247-263 (1997), the teachings of which are herebyincorporated by reference in their entirety).

After 68 hours of the 72 hour culture period, [³H]-thymidine (18.5 microBq) was added to culture media. After 4 hours of incubation with[³H]-thymidine, cells were harvested onto glass fiber filter mats by aTOMTEC harvester. Cell proliferation (also referred to as aproliferative response) was defined as the amount of [³H] thymidineincorporation into DNA over the 4 hour period as measured by liquidscintillation counting in a 1205 BETAPLATE counter. The BETAPLATEcounter had an efficiency of more than 50% for ³H. Data were expressedas counts per minute (cpm). Counts per minute was calculated as the cpmof mitogen-stimulated cultures minus the cpm of cultures withoutmitogen. The counts per minute were expressed as the mean ±SEM.

Results:

As shown in FIG. 1, the in vitro addition of nutrient mixture to invitro cultured splenocytes obtained from aced mice significantlyincreased the proliferative response of T cells to the mitogen ConA.Splenocytes obtained from aged mice and incubated in the nutrientmixture proliferated 5 fold more in response to ConA stimulation (at 1/1dilution) than splenocytes cultured in the absence of the nutrientmixture (control). An increased proliferation of T-lymphocytes inresponse to Con A indicated a measure of improvement in the immuneresponse.

The effect of the nutrient mixture on splenocyte proliferation and,thus, the immune response of cells was dose dependent. As the nutrientmixture was diluted, the proliferative response of splenocytes decreased(FIG. 1). Since proliferation of T cells in response to ConA has beenshown to be significantly reduced in aged mice and humans (Miller, R.A., Exp. Gerontol 29:21-35 (1994), the teachings of which areincorporated herein in their entirety), these results show that thenutrient mixture can significantly improve the immune response in theaged mammal.

EXAMPLE 2 Feeding the Nutrient Mixture to aged Mice SignificantlyImproved T Cell Mediated Function

Materials and Methods

Specific Pathogen-Free young male (5 months old) and aged male (24months old) C57BL/6NCrlBR mice were randomly assigned to a dietcontaining the National Research Council (NRC)-recommended level of allnutrients (control diet) or the control diet supplemented with thenutrient mixture. The composition of the control diet was based on AIN93 (Reeves, P.G., et al, J. Nutri 123: 1939-1951 (1993), the teachingsof which are hereby incorporated by reference in its entirety). Thecomponents and the concentration of components in the control diet anddiet supplemented with the nutrient mixture are shown in Table 4. Theconcentration of the components in the nutrient mixture fed to mice arelisted in Table 4.

The average caloric intake of a human is about 2100 Kcal per day(Recommended Daily Allowance, RDA). The average caloric intake of amouse is about 13.84 Kcal per day. The 13.84 Kcal per day caloric intakeis equivalent to about 3.8 g of food in a mouse diet per day. (Meydani,S. N., et al., J. Nutr. 121:547-555 (1991), the teachings of which areincorporated herein in their entirety). For example, in a human theamount of Vitamin E in a formula is 200 International Units (IU) (SeeTable 4) for a 2100 Kcal diet. To calculate the “mouse equivalent” doseor the amount to be added to a mouse diet to give a comparable amount ofVitamin E based on the 13.84 Kcal per daily caloric intake of a mousethe following calculations were employed:$\frac{200\quad {IU}\quad {Vitamin}\quad E \times 13.84\quad {Kcal}}{2100\quad {Kcal}} = {1.318\quad {IU}\quad {Vitamin}\quad E\quad {for}\quad a\quad 13.84\quad {Kcal}\quad {diet}\quad {of}\quad a\quad {mouse}}$

Since 13.84 Kcal of an average caloric intake in the mouse is equivalentto about 3.8 grams of a mouse diet (also referred to as food), theamount of Vitamin E added per kg (1000g) of mouse food was calculated asfollows:$\frac{1.318\quad {IU}\quad {Vitamin}\quad E \times 1000\quad g}{3.8\quad g} = {347\quad {IU}\quad {Vitamin}\quad E\quad {per}\quad {kg}\quad {of}\quad {food}}$

Employing the above strategy, the nutrient mixture was preparedcontaining the concentrations of each component as listed in Table 4. Anamount of the mixture was admixed with and stirred into the control dietto achieve the concentrations listed in Table 4. Food and water wereprovided ad libitum to the mice.

TABLE 4 Components in a Human Diet, Mouse Diet and Diet Fed to Mice inIn vivo Experiments the Nutrient Mixture. Control Diet Supplemented Withthe Nutrient Mixture Units: IU/kg Human Mouse Control Diet foodComponent Units/day Units/kg diet Units/kg diet or mg/kg food Vitamin E200 IU 347 IU* 30 IU 377 IU Vitamin B6 5.0 mg 8.6 mg 7.0 mg 15.67 mgConjugated 100 mg 174 mg 0 174.00 mg linoleic acid Glutathione 25 mg 44mg 0 44.0 mg Vitamin C 500 mg 868 μg 0 868.00 mg Folic Acid 500 μg 87 μg2.0 mg 2.87 mg Zinc 20 mg 34.8 mg 57.8 mg 92.60 mg Selenium 50 μg 87 μg358.7 mg 358.787 mg Vitamin D 10 μg 17.4 μg 0.0173 mg 0.0347 mg Copper1.4 μg 2.4 μg 10.5 mg 10.50243 mg Vitamin B12 1 μg 1.74 μg 0.025 mg0.02674 mg *dl-α-tocopheryl acetate

Mice were randomly divided into three treatment groups. Group I mice(n=12)were young mice (5 months of age) fed a control diet. Group IImice (n=12) were aged mice (24 months old) who were fed a control dietfor 4.5 weeks. Group II mice are also referred to as aged control mice.Group III mice (n=12) were aged mice (24 months) who were fed a controldiet supplemented with the nutrient mixture. Group III mice were alsoreferred to as aged nutrient mice. Group II and III aged mice were fedeither the control or the diet supplemented with the nutrient mixture,respectively, for 4 ½weeks.

Mice were individually housed in filtered cages in an environmentallycontrolled atmosphere (temperature 23° C., 45% relative humidity) withfifteen air changes of 100% fresh-filtered air per hour and a 12/12-hourlight-dark cycle. Mice were weighed weekly.

Food intake was recorded daily. Food intake was calculated bysubtracting the weight of left over food in each cage (one mouse percage) from the food provided. Specialized food containers, whichrestrict food spillage, were used to reduce the loss of food due todigging.

To harvest splenocytes mice were euthanized by CO₂ asphyxiation andspleens excised aseptically. Single splenocyte cell suspensions wereprepared and cultured for lymphocyte proliferation as described inExample 1 using established procedures (See, for example, Wu, et al., J.Nutr. 128:193-197 (1998), the teachings of which are hereby incorporatedby reference in their entirety). Details of culture conditions weresimilar to those described in Example 1, except the nutrient mixture wasnot added to in vitro cultures.

Results:

As shown in FIG. 2, there was no difference in weight gain between thetreatment groups.

Mice in all the treatment groups consumed about 3.8 g of food per day.There was no difference in food intake between aged mice fed a controldiet or a supplemented diet or young mice fed a control diet.

Based on the daily food intake of about 3.8 g per day, Table 5 depictsabout the concentration of each nutrient consumed by mice in each of thetreatment groups.

TABLE 5 Dietary Intake of Nutrients. Units (mg) Units (mg) ComponentGroup III Groups I and Group II Vitamin E* 1.4326* 0.114* Vitamin B60.059 0.0266 Conjugated linoleic acid 0.661 0 Glutathione 0.167 0Vitamin C 3.298 0 Folic Acid 0.011 0.0076 Zinc 0.352 0.21964 Selenium1.3634 1.3631 Vitamin D 0.000132 0.00007 Copper 0.03991 0.03990 VitaminB12 0.0001016 0.000095 *dl-α-tocopheryl acetate

FIGS. 3 and 4 show the proliferative response of splenocytes to varyingconcentrations of the T cell mitogens ConA and PHA, respectively. Valuesare expressed as the mean ±SEM for twelve individual determinations. Aproliferative response was depicted as the counts per minute (CPM) in invitro assays as described in Example 1.

Aged mice fed the control diet had a significantly lower proliferativeresponse to the T cell mitogens ConA (FIG. 3) and PHA (FIG. 4) comparedto young mice fed the control diet. Whereas the proliferative responsein splenocytes isolated from aged mice fed a diet supplemented with thenutrient mixture was significantly greater than the proliferativeresponse of splenocytes isolated from aged mice fed a control diet(FIGS. 3 and 4). An improvement in in vitro proliferation response ofsplenocytes to T-cell mitogens indicates an improvement (Beharka, A., etal., Methods in Enzymology 282:247-263 (1997), the teachings of whichare hereby incorporated by reference in their entirety).

These in vitro and in vivo data show that the nutrient mixture cansignificantly improve T cell-mediated function in the aged mice. Tcell-mediated functions can be functions that are dependent onphysiological properties of T-cells, for example, the ability of T-cellsto proliferate in response to T-cell mitogens, to produce cytokines, theability to fight viral infections, the ability to fight bacterialinfections, or the ability to attack or fight tumors. (“FundamentalImmunology”, ed. W. E. Paul, Raven Press, NY (1989), the teachings ofwhich are hereby incorporated by reference in their entirety). The mouseis an excellent model system for evaluating the effects of nutrients onimmune responses in mammals, including humans. For example, vitamin Esupplementation to old C576/6NCrlBr mice increased T cell function,vitamin E supplementation to the elderly human improved similarimmunological responses (e.g. T-cell mediated funtion) (Meydani, et al.,Mech. Aging and Develop. 34:191-201 (1986); Meydani, S. N., et al., Am.J. Clin. Nutri. 53:1275-1280 (1991), the teachings of both of which arehereby incorporated by reference in their entirety). Thus, these datastrongly suggest that supplementing humans with the nutrient mixture ofthe invention can significantly improve immune responses in humans.

T cell-mediated function decreases with aging and contributes to theincreased incidence of infectious and neoplastic diseases in the agedmammal. Thus, aged mammals can benefit from consumption of this nutrientmixture to stimulate T-cell function thereby decreasing the incidenceand severity of infectious and neoplastic diseases in the aged mammal.

When a foreign invader, such as bacteria or a virus enters the body of amammal (or human) the number to T-cells in the body proliferate tocombat the foreign invader. When lymphocytes encounter the foreigninvader, they proliferate, a phenomenon referred to as colonalexpansion. Colonal expansion enables lymphocytes to duplicate therebyproducing many similar lymphocytes in a short period of time to overcomethe foreign invader and rid the foreign invader from the body. Thus, theability of lymphocytes (e.g., T-lymphocytes) to proliferate in responseto an antigen or a mitogen (e.g. ConA, PHA) in vitro was used to assessthe ability of lymphocytes to proliferate. Lymphocytes obtained from anaging mammal, in particular T-lymphocytes, lose their ability toproliferate and, thus, the ability to combat infection in the agedmammal (e.g. rodents, primates, dogs, humans) is decreased. Thisdecreased ability to combat infection is a characteristic of lymphocytesin aged mammals. Thus, the ability of the nutrient mixture of theinvention to improve the ability of T-lymphocytes to proliferate is asignificant advantage for the elderly population to combat diseaseincluding, for example, infections and immune compromised states such asneoplastic disease.

Equivalents

While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the spirit and scope of theinvention as defined by the appended claims.

What is claimed is:
 1. A dietary supplement, comprising: a) Vitamin B6;b) Vitamin E present in a range of between about 10 and about 267milligrams per milligram of Vitamin B6; and c) conjugated linoleic acidpresent in a range of between about 17 milligrams and about 100milligrams per milligram of Vitamin B6.
 2. The dietary supplement ofclaim 1, further including: a) Vitamin C; b) folic acid; c) zinc; d)selenium; e) Vitamin D; f) copper; g) Vitamin B12; and h) glutathione.3. The dietary supplement of claim 2, wherein: a) the amount of VitaminC present is in a range of between about 17 milligrams and about 200milligrams per milligram of Vitamin B6; b) the amount of folic acidpresent is in a range of between about 0.05 milligrams and about 0.2milligrams per milligram of Vitamin B6; c) the amount of zinc present isin a range of between about 1.67 milligrams and about 10 milligrams permilligram of Vitamin B6; d) the amount of selenium present is in a rangeof between about 0.005 milligrams and about 0.02 milligrams permilligram of Vitamin B6; e) the amount of Vitamin D present is in arange of between about 0.0008 milligrams and about 0.005 milligrams permilligram of Vitamin B6; f) the amount of copper present is in a rangeof between about 0.00008 milligrams and about 0.0007 milligrams permilligram of Vitamin B6; g) the amount of Vitamin B12 present is in arange of between about 0.0002 milligrams and about 0.001 milligrams permilligram of Vitamin B6; and h) the amount of glutathione is in a rangeof between about 4 milligrams and about 33 milligrams per milligram ofVitamin
 6. 4. A dietary supplement, comprising: a) Vitamin B6; b)Vitamin E present in a range of between about 60 and about 800International Units; and c) conjugated linoleic acid present in a rangeof between about 17 milligrams and about 100 milligram per milligram ofVitamin B6.
 5. The dietary supplement of claim 4, wherein the amount ofVitamin B6 present is in a range of between about 3 milligrams and about6 milligrams.
 6. The dietary supplement comprising: a) Vitamin E presentin a range between about 60 and about 800 International Units; b)Vitamin B6 present in a range between about 3 milligrams and about 6milligrams; and c) conjugated linoleic acid present in a range betweenabout 100 milligrams and about 300 milligrams.
 7. The dietary supplementof claim 6, wherein the dietary supplement further includes glutathionein an amount in a range of between about 25 milligrams and about 100milligrams.
 8. The dietary supplement of claim 7, further including: a)about 100-600 mg of Vitamin C; b) about 300-600 μg of folic acid; c)about 10-30 mg of zinc; d) about 30-60 μg of selenium; e) about 5-15 μgof Vitamin D; f) about 0.5-2 μg of copper; and g) about 1-3 μg ofVitamin B12.
 9. A dietary supplement, comprising: a) about 60-800 IU ofVitamin E; b) about 3-6 mg of Vitamin B6; c) about 100-300 mg ofconjugated linoleic acid; and d) about 25-100 mg of glutathione.
 10. Themethod of stimulating the immune system of an individual, comprising thesteps of: a) formulating the dietary supplement that includes Vitamin E,Vitamin B6 and conjugated linoleic acid; and b) administering thedietary supplement to the individual.
 11. The method of claim 10,wherein the dietary supplement is administered to an individual that isin a range of between about 45 and about 65 years of age.
 12. A methodof claim 10, wherein the dietary supplement is administered to anindividual that is greater than about 65 years of age.
 13. The method ofclaim 10, wherein the dietary supplement further includes glutathione.14. A method of stimulating proliferation of a lymphocyte, comprisingthe steps of: a) formulating a dietary supplement that includes VitaminE, Vitamin B6, and conjugated linoleic acid; and b) administering thedietary supplement to the lymphocyte.
 15. The method of claim 14,wherein the lymphocyte is a T-cell lymphocyte.
 16. The method of claim14, wherein the dietary supplement further includes glutathione.
 17. Amethod of stimulating the immune system of an individual, comprising thesteps of: a) formulating a dietary supplement that includes about 60-800IU of Vitamin E; about 3-6 mg of Vitamin B6; and about 100-300 mg ofconjugated linoleic acid; and b) administering the dietary supplement tothe individual.
 18. The method of claim 17, wherein the dietarysupplement is administered to an individual that is middle-aged.
 19. Themethod of claim 17, wherein the dietary supplement is administered to anindividual that is elderly.
 20. A method of stimulating the immunesystem of an individual, comprising the steps of: a) formulating adietary supplement that includes about 60-800 IU of Vitamin E; about 3-6mg of Vitamin B6; about 100-300 mg of conjugated linoleic acid; andabout 25-100 mg of glutathione; and b) administering the dietarysupplement to the individual.
 21. The method of claim 20, wherein Thedietary supplement is administered to an individual that is middle-aged.22. The method of claim 20, wherein the dietary supplement isadministered to an individual that is elderly.
 23. A method ofstimulating the immune system of an individual, comprising the steps of:a) formulating a dietary supplement comprising about 60-800 IU ofVitamin E; about 3-6 mg of Vitamin B6; about 100-300 mg of conjugatedlinoleic acid; about 25-100 mg of glutathione; about 100-600 mg ofVitamin C; about 300-600 μg of folic acid; about 10-30 mg of zinc; about30-60 μg of selenium; about 5-15 μg of Vitamin D; about 0.5-2 μg ofcopper, and about 1-3 μg of Vitamin B12; and b) administering thedietary supplement to the individual.
 24. The method of claim 23,wherein the dietary supplement is administered to an individual that ismiddle-aged.
 25. The method of claim 23, wherein the dietary supplementis administered to an individual that is elderly.
 26. A method ofstimulating proliferation of a lymphocyte, comprising the steps of: a)formulating a dietary supplement that includes about 60-800 IU ofVitamin E; about 3-6 mg of Vitamin B6; and about 100-300 mg ofconjugated linoleic acid; and b) administering the dietary supplement tothe lymphocyte.
 27. The method of claim 26, wherein the dietarysupplement is administered to a T-cell lymphocyte.
 28. A method ofstimulating proliferation of a lymphocyte, comprising the steps of: a)formulating a dietary supplement that includes about 60-800 IU ofVitamin E; about 3-6 mg of Vitamin B6; about 100-300 mg of conjugatedlinoleic acid; and about 25-100 mg of glutathione; and b) administeringthe dietary supplement to the lymphocyte.
 29. The method of claim 28,wherein the dietary supplement is administered to a T-cell lymphocyte.30. A method of stimulating proliferation of a lymphocyte, comprisingthe steps of: a) formulating a dietary supplement comprising about60-800 IU of Vitamin E; about 3-6 mg of Vitamin B6; about 100-300 mg ofconjugated linoleic acid; about 25-100 mg of glutathione; about 100-600mg of Vitamin C; about 300-600 μg of folic acid; about 10-30 mg of zinc;about 30-60 μg of selenium; about 5-15 μg of Vitamin D; about 0.5-2 μgof copper, and about 1-3 μg of Vitamin B12; and b) administering thedietary supplement to the lymphocyte.
 31. The method of claim 30,wherein the lymphocyte is a T-cell lymphocyte.